U.S. patent application number 11/955080 was filed with the patent office on 2008-07-31 for degradation-resistant fibrinogen sealants.
Invention is credited to David H. Farrell.
Application Number | 20080181878 11/955080 |
Document ID | / |
Family ID | 39668256 |
Filed Date | 2008-07-31 |
United States Patent
Application |
20080181878 |
Kind Code |
A1 |
Farrell; David H. |
July 31, 2008 |
DEGRADATION-RESISTANT FIBRINOGEN SEALANTS
Abstract
Provided are degradation-resistant fibrinogen sealants
comprising a first composition comprising one or more of fibrinogen
.gamma.A/.gamma.' heterodimers and/or fibrinogen .gamma.'/.gamma.'
homodimers and a second composition comprising thrombin and,
optionally, degradation-resistant fibrinogen sealants disclosed
herein may further comprise Factor XIII and calcium.
Degradation-resistant fibrinogen sealants are suitable for the
treatment of trauma, particularly vascular trauma.
Inventors: |
Farrell; David H.;
(Tualatin, OR) |
Correspondence
Address: |
DANN, DORFMAN, HERRELL & SKILLMAN
1601 MARKET STREET, SUITE 2400
PHILADELPHIA
PA
19103-2307
US
|
Family ID: |
39668256 |
Appl. No.: |
11/955080 |
Filed: |
December 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60874310 |
Dec 12, 2006 |
|
|
|
Current U.S.
Class: |
424/94.64 |
Current CPC
Class: |
A61K 38/4833 20130101;
A61L 24/106 20130101; A61K 38/363 20130101; A61K 38/45 20130101;
A61K 38/45 20130101; A61K 38/4833 20130101; A61P 7/00 20180101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61P 9/00 20180101; A61K 38/363 20130101 |
Class at
Publication: |
424/94.64 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61P 7/00 20060101 A61P007/00; A61P 9/00 20060101
A61P009/00 |
Goverment Interests
[0002] Pursuant to 35 U.S.C. .sctn.202(c) it is acknowledged that
the U.S. Government has certain rights in the invention described,
which was made in part with funds from the National Institutes of
Health, Grant Number NIH/NHLBI 1 R29 HL53997.
Claims
1. A degradation-resistant fibrinogen sealant, comprising a first
composition and a second composition; wherein said first
composition comprises at least one fibrinogen dimer selected from
the group consisting of fibrinogen .gamma.A/.gamma.' heterodimer
and fibrinogen .gamma.'/.gamma.' homodimer; and wherein said second
composition comprises thrombin.
2. The degradation-resistant fibrinogen sealant of claim 1 wherein
each of said fibrinogen dimers is present at a concentration of
between about 5 mg/ml to about 200 mg/ml.
3. The degradation-resistant fibrinogen sealant of claim 1 wherein
each of said fibrinogen dimers is present at a concentration of
between about 10 mg/ml to about 200 mg/ml.
4. The degradation-resistant fibrinogen sealant of claim 1 wherein
each of said fibrinogen dimers is present at a concentration of
between about 25 mg/ml to about 150 mg/ml.
5. The degradation-resistant fibrinogen sealant of claim 1 wherein
each of said fibrinogen dimers is present at a concentration of
between about 40 mg/ml to about 130 mg/ml.
6. The degradation-resistant fibrinogen sealant of claim 1 wherein
each of fibrinogen dimers is present at a concentration of between
about 65 mg/ml and about 115 mg/ml.
7. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition is substantially pure of any other
fibrinogen.
8. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition comprises fibrinogen .gamma.A/.gamma.'.
9. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first com position comprises fibrinogen .gamma.'/.gamma.'.
10. The degradation-resistant fibrinogen sealant of claim 1 wherein
said fibrinogen .gamma.A/.gamma.' is present at between about 5%
and about 90% of the total fibrinogen.
11. The degradation-resistant fibrinogen sealant of claim 1 wherein
said fibrinogen .gamma.A/.gamma.' is present at between about 10%
and about 80% of the total fibrinogen.
12. The degradation-resistant fibrinogen sealant of claim 1 wherein
said fibrinogen .gamma.A/.gamma.' is present at between about 20%
and about 70% of the total fibrinogen.
13. The degradation-resistant fibrinogen sealant of claim 1 wherein
said fibrinogen .gamma.A/.gamma.' is present at a concentration
selected from the group consisting of about 30%, 40%, 50%, and 60%
of the total fibrinogen.
14. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition further comprises Factor XIII.
15. The degradation-resistant fibrinogen sealant of claim 14
wherein said Factor XIII is present at a concentration of from
between about 10 U/ml and about 80 U/ml.
16. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition has a pH of between about pH 5.0 and about
pH 9.0.
17. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition has a pH of between about 5.5 and about pH
8.5.
18. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition has a pH of between about pH 6.0 and about
pH 8.0.
19. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition has a pH of between about pH 6.5 and about
pH 7.5.
20. The degradation-resistant fibrinogen sealant of claims 1
wherein said first composition has a pH of between about pH 6.7 and
about pH 7.2.
21. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition further comprises at least one
pharmaceutically acceptable carrier.
22. The degradation-resistant fibrinogen sealant of claim 1 wherein
said second composition further comprises at least one
pharmaceutically acceptable carrier.
23. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition further comprises at least one compound
selected from the group consisting of tranexamic acid, arginine
hydrochloride, glycine, sodium chloride, sodium citrate, and
calcium chloride.
24. The degradation-resistant fibrinogen sealant of claims 1
wherein said thrombin is present at a concentration of between
about 4 IU/ml and about 1000 IU/ml.
25. The degradation-resistant fibrinogen sealant of claim 1 wherein
said thrombin is present at a concentration of between about 10
IU/ml and about 150 IU/ml.
26. The degradation-resistant fibrinogen sealant of claim 1 wherein
said thrombin is present at a concentration of between about 15
IU/ml and about 120 IU/ml.
27. The degradation-resistant fibrinogen sealant of claim 1 wherein
said thrombin is present at a concentration selected from the group
consisting of about 25 IU/ml, 50 IU/ml, 75 IU/ml, and 100
IU/ml.
28. The degradation-resistant fibrinogen sealant of claim 1 wherein
said second composition further comprises calcium.
29. The degradation-resistant fibrinogen sealant of claim 28
wherein said calcium is present at a concentration of between about
10 mM and about 70 mM.
30. The degradation-resistant fibrinogen sealant of claim 28
wherein said calcium is present at a concentration of between about
20 mM and about 60 mM.
31. The degradation-resistant fibrinogen sealant of claim 28
wherein said calcium is present at a concentration of between about
30 mM and about 50 mM.
32. The degradation-resistant fibrinogen sealant of claim 1 further
comprising at least one compound selected from the group consisting
of human albumin, mannitol, and sodium acetate.
33. The degradation-resistant fibrinogen sealant of claim 1 wherein
the pH of said second composition is between about pH 5.0 and about
pH 9.0.
34. The degradation-resistant fibrinogen sealant of claim 1 wherein
the pH of said second composition is between about pH 5.5 and about
pH 8.5.
35. The degradation-resistant fibrinogen sealant of claim 1 wherein
the pH of said second composition is between about pH 6.0 and about
pH 8.0.
36. The degradation-resistant fibrinogen sealant of claim 1 wherein
the pH of said second composition is between about pH 6.5 and about
pH 7.5.
37. The degradation-resistant fibrinogen sealant of claim 1 wherein
the pH of said second composition is between about pH 6.8 and about
pH 7.2.
38. The degradation-resistant fibrinogen sealant of claim 1 further
comprising at least one fibrin monomer selected from the group
consisting of fibrin I and fibrin II monomers.
39. The degradation-resistant fibrinogen sealant of claim 1 wherein
said first composition further comprises fibrinogen
.gamma.A/.gamma.A.
40. A method for the treatment of vascular trauma in a patient,
said method comprising the step of administering to said patient
the degradation-resistant fibrinogen sealant of claim 1.
41. A kit comprising the degradation-resistant fibrinogen sealant
of claim 1 and a syringe.
Description
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 60/874,310,
filed on Dec. 12, 2006. The foregoing application is incorporated
by reference herein.
FIELD OF THE INVENTION
[0003] The present disclosure relates generally to the treatment of
wounds, particularly vascular wounds. More specifically, the
present disclosure provides degradation-resistant fibrinogen
sealants having a first composition comprising fibrinogen
.gamma.A/.gamma.' heterodimers and/or fibrinogen .gamma.'/.gamma.'
homodimers and a second composition comprising thrombin.
Optionally, degradation-resistant fibrinogen sealants disclosed
herein may further comprise Factor XIII and calcium.
BACKGROUND OF THE INVENTION
[0004] Several publications and patent documents are cited
throughout the specification in order to describe the state of the
art to which this invention pertains. Each of these citations is
incorporated herein by reference as though set forth in full.
[0005] Blood clot formation occurs through the conversion of
fibrinogen by thrombin and Factor XIIIa to form a cross-linked
fibrin polymer. Fibrinogen is a 340,000-Da dimeric glycoprotein
composed of six disulphide-linked polypeptide chains: two
A.alpha.(M.sub.r=65,000), two B.beta.(M.sub.r=56,000), and two
.gamma.(M.sub.r=47,000). Fibrinogen is converted to fibrin through
limited proteolysis by thrombin, which exposes polymerization sites
in fibrinogen (Kudryk et al. (1974) J. Biol. Chem. 249:3322-3325).
The fibrin monomers spontaneously associate with each other to form
the web-like fibrin clot (Blomback (1996) Thromb. Res.
83:1-75).
[0006] Factor XIIIa is a plasma transglutaminase that strengthens
the fibrin clot by forming covalent bonds between adjacent fibrin
monomers (Lorand et al. (1993) Methods Enzymol. 222:22-35). Plasma
Factor XIII is a 320,000-Da tetrameric protein composed of two
polypeptide a chains (M.sub.r=83,000) and two polypeptide b chains
(M.sub.r=80,000; Schwartz et al. (1973) J. Biol. Chem.
248:1395-1407). Factor XIII normally circulates as an inactive
proenzyme until it is activated by thrombin cleavage of a 4000-Da
activation peptide from each a subunit, which is followed by the
dissociation of the b subunits. Activated factor XIII, or XIIIa,
catalyzes the formation of .gamma.-glutamyl-.epsilon.-lysine bonds
between polypeptide chains in fibrin (Chen et al. (1969) Proc.
Natl. Acad. Sci. U.S.A. 63:420-427). These cross-links strengthen
the fibrin clot (Lorand (1972) Ann. N. Y. Acad. Sci. 202:6-30) and
increase its resistance to lysis (Gaffney and Whitaker (1979)
Thromb. Res. 14:85-94; Reed et al. (1992) Thromb. Haemostasis
68:315-320; Siebenlist and Mosesson (1994) J. Biol. Chem.
269:28414-28419).
[0007] Trauma is the leading cause of death for people between the
ages of 1 and 44 in the United States (Bonne et al., eds. "Reducing
the Burden of Injury: Advancing Prevention and Treatment."
Committee on Injury Prevention and Control, Institute of Medicine
(Washington, D.C., National Academy Press, 1999). The majority of
deaths that occur during the first 48 hours following a traumatic
event are the result of uncontrolled bleeding (Sauaia et al. (1995)
"Epidemiology of Trauma Deaths: A Reassessment" J. Trauma
38:185-193). A common result of traumatic injury is disseminated
intravascular coagulation (DIC), in which the activation of
fibrinolytic enzymes causes the clot to dissolve. Massive
hemorrhage can be resistant even to high doses of recombinant
factor VIIa. The primary treatment of such injuries is therefore
surgical repair, which is often aided by the use of fibrin sealants
to stop hemorrhage. Fibrin sealants, such as BERIPLAST-P.TM.
(Aventis-Behring), CROSSEAL.TM. (Johnson & Johnson), and
TISSEEL.TM. (Baxter) may be applied during surgery from a
two-syringe system. One syringe contains the fibrin precursor
protein, fibrinogen, and the other syringe contains the clotting
factor thrombin. These two components may be forced into a mixing
chamber and act much like a two-part epoxy resin in which
fibrinogen serves as the resin and thrombin serves as the catalyst.
The mixture coagulates within minutes and stops bleeding from the
wound site.
[0008] Fibrinolytic enzymes that are activated in DIC can, however,
digest the applied fibrin sealant, resulting in re-bleeding of the
wound even after initial control of hemorrhage. Furthermore,
inhibitors of the fibrinolytic enzymes that are sometimes added to
fibrin sealant, such as aprotinin, can be immunogenic and cause
anaphylactic reactions. Therefore, there is still a need for
degradation resistant fibrin sealants which avoid these
drawbacks.
SUMMARY OF THE INVENTION
[0009] The present disclosure fulfills these and other related
needs by providing degradation-resistant fibrinogen sealants that
may be used in a wide variety of surgical applications including,
for example, open surgery, trauma surgery, plastic surgery, general
surgery, dental surgery, minimally invasive surgery, endoscopy, and
microsurgery. Degradation-resistant fibrinogen sealants disclosed
herein employ one or more fibrinogen dimers selected from a
.gamma.A/.gamma.' heterodimer and a .gamma.'/.gamma.' homodimer in
combination with thrombin. Fibrinogen sealants may, optionally,
also include one or more of Factor XIII and/or calcium.
[0010] Fibrinogen sealants are advantageously formulated as two
separate compositions. A first composition contains one or more
fibrinogen dimer (i.e. a fibrinogen .gamma.A/.gamma.' heterodimer
and/or fibrinogen .gamma.'/.gamma.' homodimer) and a second
composition containing thrombin. The first composition may
additionally contain Factor XIII. The second composition may
additionally contain calcium (e.g., CaCl.sub.2). When the two
solutions are mixed, at the time of administration to a trauma
patient in need thereof, the thrombin in the second composition
converts the fibrinogen dimers to fibrin. In those embodiments
further employing Factor XIII in the first composition, thrombin
also converts the zymogen (inactive) form of Factor XIII to the
active form that, in the presence of calcium, covalently
cross-links the polymerized fibrinogen molecules.
[0011] In a particular embodiment of the instant invention, the
compositions comprise at least one pharmaceutically acceptable
carrier. Within certain aspects of these embodiments, the first
composition may comprise one or more additional components selected
from the group consisting of tranexamic acid, arginine
hydrochloride, glycine, sodium chloride, sodium citrate, and
calcium chloride.
[0012] The fibrinogen .gamma.A/.gamma.' heterodimer and/or a
.gamma./.gamma.' homodimer may be present in the first composition
at a concentration of between about 5 mg/ml to about 200 mg/ml,
between about 10 mg/ml to about 200 mg/ml, between about 25 mg/ml
to about 150 mg/ml, or between about 40 mg/ml to about 130 mg/ml.
In a particular embodiment, a fibrinogen .gamma.A/.gamma.'
heterodimer and/or a .gamma.'/.gamma.' homodimer is present in the
first composition at a concentration of between about 65 mg/ml and
about 115 mg/ml. In another embodiment, the fibrinogen
.gamma.A/.gamma.' heterodimer and/or a .gamma.'/.gamma.' homodimer
is substantially pure. The fibrinogen .gamma.A/.gamma.' heterodimer
and/or a .gamma.'/.gamma.' homodimer may be the only fibrinogen
present in the compositions of the instant degradation-resistant
fibrinogen sealants.
[0013] In yet another embodiment, fibrinogen .gamma.'/.gamma.' may
also be present in the first compositions as described above.
Fibrinogen .gamma.'/.gamma.' may comprise between about 5% and
about 90% of the total fibrinogen in a first composition. In a
particular embodiment, .gamma.'/.gamma.' fibrinogen is present in
the first composition at between about 10% and about 80% of the
total fibrinogen, at between about 20% and about 70% of the total
fibrinogen, or at about 30%, 40%, 50%, or 60% of the total
fibrinogen.
[0014] Within those aspects wherein the first composition further
comprises Factor XIII, it may be present at a concentration of from
between about 10 U/ml and about 80 U/ml.
[0015] The pH of the first composition may be between about pH 5.0
and about pH 9.0, between about pH 5.5 and about pH 8.5, between
about pH 6.0 and about pH 8.0, or between about pH 6.5 and about pH
7.5. In a particular embodiment, the pH of the first composition is
between about pH 6.7 and about pH 7.2.
[0016] As indicated above, second compositions comprise thrombin.
Thrombin is usually present in second compositions at a
concentration of between about 4 IU/ml and about 1000 IU/ml,
between about 10 IU/ml and about 150 IU/ml, or between about 15
IU/ml and about 120 IU/ml. Particularly, thrombin may be present in
second compositions at a concentration of 25 IU/ml, 50 IU/ml, 75
IU/ml, or 100 IU/ml.
[0017] In those embodiments wherein calcium is present in the
second composition, the concentration of calcium is typically
between about 1 mM and about 70 mM, more typically between about 20
mM and about 60 mM, most typically between about 30 mM and about 50
mM. In some embodiments, a second solution may also contain human
albumin, mannitol, and/or sodium acetate.
[0018] The pH of the second composition is usually between pH 5.0
to pH 9.0, between pH 5.5 to pH 8.5, between pH 6.0 to pH 8.0,
between pH 6.5 to pH 7.5, or between pH 6.8 to pH 7.2.
[0019] Within certain aspects, degradation-resistant fibrinogen
sealants disclosed herein may further employ one or more fibrin I
and/or fibrin II monomer(s). Thus, for example, fibrin I monomers
and/or fibrin II monomers may be prepared in advance of sealant
application from fibrinogen using, for example, a proteolytic
enzyme such as thrombin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a graph representing the mean arterial pressure in
pigs following aortic injury and treatment with CROSSEAL.TM.
fibrinogen sealant, a degradation-resistant .gamma.A/.gamma.'
fibrin sealant of the present invention, or albumin.
[0021] FIG. 2 is a graph representing percentage of clot lysis as a
function of the percent of .gamma.A/.gamma.' fibrinogen in the
clot.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present disclosure is predicated on the observation that
degradation-resistant fibrinogen sealants may be prepared from a
combination of one or more .gamma.A/.gamma.' fibrinogen
heterodimers and/or .gamma.'/.gamma.' fibrinogen homodimers. Thus,
the present disclosure provides degradation-resistant fibrinogen
sealants employing a first composition comprising one or more
.gamma.A/.gamma.' fibrinogen heterodimer and/or .gamma.'/.gamma.'
fibrinogen homodimer and optionally further comprising, without
limitation, at least one pharmaceutically acceptable carrier,
antibiotic, stabilizer, Factor XIII, tranexamic acid, arginine
hydrochloride, glycine, sodium chloride, sodium citrate, and/or
calcium chloride and a second composition comprising thrombin and
optionally further comprising, without limitation, at least one
pharmaceutically acceptable carrier, antibiotic, stabilizer, and/or
calcium.
[0023] Fibrinogen is a 340,000-Da dimeric glycoprotein composed of
six disulphide-linked polypeptide chains: two
A.alpha.(M.sub.r=65,000), two B.beta.(M.sub.r=56,000), and two
.gamma.(M.sub.r=47,000). "A" and "B" represent two small amino
terminal peptides, known as fibrinopeptide A and fibrinopeptide B,
respectively. The formation of insoluble fibrin clots (e.g.,
crosslinked fibrin II polymer) is believed to begin with fibrinogen
being converted by thrombin to fibrin I monomer. This conversion
involves thrombin-mediated cleavage of the 16 amino acid
fibrinopeptide A from each of the two A.alpha. chains of
fibrinogen, producing two .alpha.-chains each with a new
N-terminus. It is believed that the fibrin I monomer can
spontaneously polymerize with other fibrin I or fibrin II
monomers.
[0024] The next step in the formation of fibrin clots is believed
to involve the conversion of fibrin I monomer to fibrin II monomer.
This step involves the thrombin-mediated cleavage of the
fibrinopeptide B from each of the two B.beta. chains of fibrin I.
Fibrin II monomers, like fibrin I monomers, can spontaneously
polymerize with other fibrin II or fibrin I monomers. Activated
Factor XIIIa covalently crosslinks adjacent fibrin II monomers in
the fibrin II polymer. Factor XIIIa is also capable of crosslinking
fibrin I monomers in a fibrin polymer.
[0025] In plasma-derived fibrinogen, there are two alternatively
spliced gamma chains, .gamma.A and .gamma.'. The .gamma.' chain
arises from alternative processing of the .gamma. chain mRNA that
leads to the translation of a polypeptide with a 20-amino acid
sequence substituted for the carboxyl-terminal four amino acids of
the .gamma.A chain (Chung and Davie (1984) Biochemistry
23:4232-4236; Fornace et al. (1984) J. Biol. Chem.
259:12826-12830). In human plasma, about 90% of the fibrinogen
present is .gamma.A/.gamma.A -fibrinogen, and the remaining 10% is
.gamma.A/.gamma.'-fibrinogen. The .gamma.' chain binds to Factor
XIII (Siebenlist et al. (1996) Biochemistry 35:10448-10453).
[0026] Both the rate of clotting and the rate of lysis are
significantly decreased in .gamma.A/.gamma.' fibrin clots as
compared to .gamma.A/.gamma.A fibrin clots (Falls and Farrell
(1997) J. Biol. Chem. 272:14251-14256). Clots made from
.gamma.A/.gamma.' fibrinogen in the presence of Factor XIII clot
more slowly and subsequently lyse more slowly. Clot stability is
enhanced further in the presence of supraphysiological
concentrations of Factor XIII. Fibrinogen can polymerize into a
clot or gel, which is able to act as a sealant, glue, hemostat, or
wound healing matrix in vitro and in vivo.
Definitions
[0027] "Nucleic acid" or a "nucleic acid molecule" as used herein
refers to any DNA or RNA molecule, either single or double stranded
and, if single stranded, the molecule of its complementary sequence
in either linear or circular form. In discussing nucleic acid
molecules, a sequence or structure of a particular nucleic acid
molecule may be described herein according to the normal convention
of providing the sequence in the 5' to 3' direction. With reference
to nucleic acids of the invention, the term "isolated nucleic acid"
is sometimes used. This term, when applied to DNA, refers to a DNA
molecule that is separated from sequences with which it is
immediately contiguous in the naturally occurring genome of the
organism in which it originated. For example, an "isolated nucleic
acid" may comprise a DNA molecule inserted into a vector, such as a
plasmid or virus vector, or integrated into the genomic DNA of a
prokaryotic or eukaryotic cell or host organism.
[0028] When applied to RNA, the term "isolated nucleic acid" refers
primarily to an RNA molecule encoded by an isolated DNA molecule as
defined above. Alternatively, the term may refer to an RNA molecule
that has been sufficiently separated from other nucleic acids with
which it would be associated in its natural state (i.e., in cells
or tissues). An "isolated nucleic acid" (either DNA or RNA) may
further represent a molecule produced directly by biological or
synthetic means and separated from other components present during
its production.
[0029] A "vector" is a replicon, such as a plasmid, cosmid, bacmid,
phage or virus, to which another genetic sequence or element
(either DNA or RNA) may be attached so as to bring about the
replication of the attached sequence or element.
[0030] An "expression operon" refers to a nucleic acid segment that
may possess transcriptional and translational control sequences,
such as promoters, enhancers, translational start signals (e.g.,
ATG or AUG codons), polyadenylation signals, terminators, and the
like, and which facilitate the expression of a polypeptide coding
sequence in a host cell or organism.
[0031] The term "substantially pure" refers to a preparation
comprising at least 50-60% by weight of a given material (e.g.,
nucleic acid, oligonucleotide, protein, etc.). More preferably, the
preparation comprises at least 75% by weight, more preferably about
90-95% by weight, and more preferably about 99% by weight of the
given compound. Purity is measured by methods appropriate for the
given compound (e.g., chromatographic methods, agarose or
polyacrylamide gel electrophoresis, HPLC analysis, and the
like).
[0032] The term "isolated protein" or "isolated and purified
protein" refers primarily to a protein produced by expression of an
isolated nucleic acid molecule of the invention. Alternatively,
this term may refer to a protein that has been sufficiently
separated from other proteins with which it would naturally be
associated, so as to exist in "substantially pure" form. "Isolated"
is not meant to exclude artificial or synthetic mixtures with other
compounds or materials, or the presence of impurities that do not
interfere with the fundamental activity, and that may be present,
for example, due to incomplete purification, or the addition of
stabilizers.
[0033] The term "gene" refers to a nucleic acid comprising an open
reading frame encoding a polypeptide, including both exon and
(optionally) intron sequences. The nucleic acid may also optionally
include non coding sequences such as promoter or enhancer
sequences. The term "intron" refers to a DNA sequence present in a
given gene that is not translated into protein and is generally
found between exons.
[0034] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of the
composition of the invention for performing a method of the
invention.
[0035] The term "antibiotics" refers to, without limitation,
.beta.-lactams (penicillins and cephalosporins), vancomycins,
bacitracins, macrolides (erythromycins), lincosamides
(clindomycin), chloramphenicols, tetracyclines (e.g.,
immunocycline, chlortetracycline, oxytetracycline, demeclocycline,
methacycline, doxycycline and minocycline), aminoglycosides (e.g.,
gentamicins, amikacins, and neomycins), amphotericins, cefazolins,
clindamycins, mupirocins, sulfonamides and trimethoprim,
rifampicins, metronidazoles, quinolones, novobiocins, polymixins
and gramicidins and the like and any salts or variants thereof.
[0036] The term "stabilizer" refers to a chemical agent (e.g.,
protein or polysaccharide) that assists to preserve or maintain the
biological structure and/or biological activity of a protein.
Examples of stabilizers include, without limitation, hydroxyethyl
starch (hetastarch), serum albumin, gelatin, collagen, recombinant
albumin, recombinant gelatin, recombinant collagen, non-oxidizing
amino acid derivatives (e.g., tryptophan derivatives, such as
N-acetyl-tryptophan), caprylates, polysorbates, amino acids, and
divalent metal cations (e.g., Zn.sup.2+), and cresols.
[0037] The term "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media and the like which may be
appropriate for the desired route of administration of the
pharmaceutical composition. The use of such media for
pharmaceutically active substances is known in the art. Except
insofar as any conventional media or agent is incompatible with the
compounds to be administered, its use in the pharmaceutical
preparation is contemplated. A "carrier" refers to, for example, a
diluent, adjuvant, excipient, auxiliary agent or vehicle with which
an active agent of the present invention is administered. Examples
of pharmaceutically acceptable carriers include, without
limitation, water, buffered saline, ethanol, polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycol and the
like), dimethyl sulfoxide (DMSO), oils, detergents, suspending
agents or suitable mixtures thereof. Suitable pharmaceutically
acceptable carriers and formulations are described in Remington's
Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton,
1995) and "Remington: The Science And Practice Of Pharmacy" by
Alfonso R. Gennaro (Lippincott Williams & Wilkins, 2005).
Preparation of Degradation-Resistant Fibrinogen Sealants
[0038] Degradation-resistant fibrinogen sealants disclosed herein
include a first composition comprising one or more of fibrinogen
.gamma.A/.gamma.' heterodimers and/or fibrinogen .gamma.'/.gamma.'
homodimers and a second composition comprising thrombin. As
indicated above, degradation-resistant fibrinogen sealants may
additionally include, in a first composition, Factor XIII and, in a
second composition, calcium (e.g., CaCl.sub.2).
Degradation-resistant fibrinogen sealants may be used in many
applications including, for example, open surgery, trauma surgery,
plastic surgery, general surgery, dental surgery, minimally
invasive surgery, endoscopy, and microsurgery.
[0039] When the first composition and the second composition are
mixed, the thrombin converts fibrinogen to fibrin. In some
embodiments, thrombin also converts the zymogen (inactive) form of
Factor XIII to the active form that, in the presence of calcium,
covalently cross-links the polymerized fibrinogen molecules.
[0040] A fibrinogen .gamma.A/.gamma.' heterodimer and/or a
fibrinogen .gamma.'/.gamma.' homodimer may be present in the first
composition at a concentration of between about 5 mg/ml to about
200 mg/ml, between about 10 mg/ml to about 200 mg/ml, between about
25 mg/ml to about 150 mg/ml, or between about 40 mg/ml to about 130
mg/ml. Particularly, a fibrinogen .gamma.A/.gamma.' heterodimer
and/or a .gamma.'/.gamma.' homodimer is present in the first
composition at a concentration of between about 65 mg/ml and about
115 mg/ml. Suitable concentrations of fibrinogen .gamma.A/.gamma.'
heterodimers and/or a fibrinogen .gamma.'/.gamma.' homodimers may
be achieved by precipitation using ethanol (EtOH) and low
temperature (Dahlstrom et al. (1992) Plast. Reconstr. Surg.
89:968-972). Other precipitation methods may also be suitably
employed for concentrating solutions comprising fibrinogen
.gamma.A/.gamma.' heterodimers and/or a fibrinogen
.gamma.'/.gamma.' homodimers (such as, e.g., glycine or ammonium
sulphate precipitation).
[0041] Fibrinogen .gamma.'/.gamma.' may comprise between about 0%
and about 100% of the total fibrinogen in a first composition,
between about 5% and about 90%, between about 10% and about 80% of
the total fibrinogen, or between about 20% and about 70% of the
total fibrinogen. In a particular embodiment, fibrinogen
.gamma.'/.gamma.' may be present in the first composition at about
30%, 40%, 50%, or 60% of the total fibrinogen. If the fibrinogen is
isolated from plasma, fibrinogen .gamma.'/.gamma.' may be present
in trace amounts.
[0042] Fibrinogen .gamma.A/.gamma.A may also be present in the
first composition. Fibrinogen .gamma.A/.gamma.A possesses
degradation resistance properties similar to unfractionated
fibrinogen. Accordingly, the addition of fibrinogen
.gamma.A/.gamma.A to the first composition at different ratios to
fibrinogen .gamma.A/.gamma.' and .gamma.'/.gamma.' modulates the
degradation resistance (e.g., an increase in the ratio of
fibrinogen .gamma.A/.gamma.A would decrease the resistance to
degradation). Fibrinogen .gamma.A/.gamma.A may comprise between
about 5% and about 90% of the total fibrinogen is a first
composition, between about 10% and about 80% of the total
fibrinogen, or between about 20% and about 70% of the total
fibrinogen. In a particular embodiment, fibrinogen
.gamma.A/.gamma.A may be present in the first composition at about
30%, 40%, 50%, or 60% of the total fibrinogen.
[0043] Fibrinogen may be derived from pooled plasma, such as pooled
human plasma. Fibrinogen may also be obtained from single-donor and
autologous sources (e.g., from blood banks). The fibrinogen can be
concentrated from the plasma by cryoprecipitation and precipitation
using various reagents including, for example, poly(ethylene
glycol), diethyl ether, ethanol, ammonium sulfate, and glycine. In
a particular embodiment, the .gamma.A/.gamma.' and
.gamma.'/.gamma.' fibrinogen are separated from .gamma.A/.gamma.A
fibrinogen. The .gamma.A/.gamma.' and .gamma.'/.gamma.' fibrinogen
may be separated, for example, by ion-exchange (Mosesson et al.
(1972) J. Biol. Chem., 247:5223-5227) or affinity chromatography
using an anti-.gamma.' antibody such as 2.G2.H9 (Lovely et al.
(2002) Thromb. Haemost., 88:26-31).
[0044] Fibrinogen may also be produced by chemical synthesis (see,
e.g., Merrifield, (1963) J. Chem. Soc. 85:2149-2154; Hunkapillar et
al., (1984) Nature 310:105-111) or by a recombinant process. For
example, recombinant fibrinogen may be produced in the body fluids
of transgenic animals as taught by WO 95/23868, which is herein
incorporated by reference in its entirety. For example, fibrinogen
may be recombinantly produced in the milk of placental mammals such
as sheep, pigs, cattle goats, rabbits, and camels.
[0045] Genetic engineering may be used to produce fibrinogen and
fibrin monomers in comparatively high yields. Heterologous
expression of fibrinogen and fibrin chains also allows the
construction of mutations that can mimic, for example, naturally
occurring fibrin variants.
[0046] Each of the three polypeptide chains of fibrinogen
(A.alpha., B.beta., and .gamma.) is coded by a separate gene.
Nucleotide sequences encoding fibrinogen, thrombin, Factor XIII, or
other genes can be constructed using any known method. For example,
nucleotide sequences can be chemically synthesized or synthesized
using polymerase chain reaction (PCR) amplification (see, e.g.,
Gelfind, "PCR Technology: Principles and Applications for DNA
Amplification" (Ed., H. A. Erlich, Stockton Press, N.Y.,1989);
"Current Protocols in Molecular Biology" Vol. 2, Ch. 15 (Eds.
Ausubel et al., John Wiley & Sons (1988); Horton et al. (1989)
Gene 77:61-68).
[0047] Nucleotide sequences can also be constructed using
recombinant DNA techniques (see, e.g., Sambrook et al., "Molecular
Cloning: A Laboratory Manual" 2.sup.nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989). Vectors containing one
or more nucleotide sequence may also be constructed. Possible
vectors include, but are not limited to, plasmids, cosmids, or
modified viruses or bacteriophages. These vectors may be used to
transfect a procaryotic or eucaryotic host cell.
[0048] The cDNAs for each of the fibrinogen chains may be prepared
and expressed in procaryotic organisms (Chung et al. (1983) Ann.
N.Y. Acad. Sci. 408:449-456; Rixen et al., (1983) Biochemistry
22:3227-3244; Chung et al. (1983) Biochemistry 22:3244-3250; and
Chung et al. (1983) Biochemistry 22:3250-3256). Each human
fibrinogen chain is typically introduced separately (Huang et al.
(1983) J. Biol. Chem. 268:8919-8926; Roy et al. (1992) J. Biol.
Chem. 267:23151-23158; Roy et al. (1991) J. Biol. Chem.,
266:4758-4763). Alternatively, human fibrinogen chains may be
introduced in combination into expression plasmids and transfected
into eukaryotic cells (Farrell et al. (1991) Biochemistry
30:9414-9420; Hartwig and Danishefsky (1991) J. Biol. Chem.
266:6578-6585; Huang et al. (1983) J. Biol. Chem. 268:8919-8926;
Roy et al. (1991) J. Biol. Chem. 266:4758-4763).
[0049] Suitable plasmids for use in expressing recombinant human
fibrinogen have been described (see, e.g., Rixen et al. (1983)
Biochemistry 22:3237-3244; Chung et al. (1983) Biochemistry
22:3244-3250; Chung et al. (1983) Biochemistry 22:3250-3256).
Recombinant fibrinogen chains may be expressed in E. coli. (see,
e.g., Bolyard and Lord (1988) Gene 66:183; Bolyard and Lord (1989)
Blood 73:1202-1206; Lord and Fowlkes (1989) Blood 73:166-171).
[0050] Eukaryotic cells carrying expression plasmids encoding
individual fibrinogen chains have been shown to synthesize the
encoded fibrinogen chains and to result in the intracellular
formation of dimeric chain molecules, e.g., A.alpha., B.beta., or
.gamma. dimers (Roy et al. (1990) J. Biol. Chem. 265:6389-6393;
Zhang and Redman (1992) J. Biol. Chem. 267:21727-21732). When
appropriate plasmids containing genes encoding all three human
fibrinogen chains are transferred into the same cell, then not only
are all three chains expressed but the polypeptide chains associate
in pairs and intact fibrinogen is secreted into the surrounding
medium (Farrell et al. (1991) Biochemistry 30:9414-9420; Roy et al.
(1991) J. Biol. Chem. 266:4758-4763; Hartwig and Danishefsky (1991)
J. Biol. Chem. 266:6578-6585). The secreted recombinant fibrinogen
is functional in forming fibrin polymers.
[0051] Fibrinogen is naturally synthesized by, for example, liver
and megakaryocyte cells. Transformed liver cells maintained in
culture are able to continue fibrinogen synthesis and secretion
(Otto et al. (1987) J. Cell Biol. 105:1067-1072; Yu et al. (1987)
Thromb. Res. 46:281-293; Alving et al. (1982) Arch. Biochem.
Biophys. 217:19). Hep G2 cells synthesize an excess of A.alpha. and
.gamma. chains over B.beta. chains, but the introduction of an
additional expression vector encoding .beta. chains resulted in the
formation of trimeric complexes (A.alpha.B.beta..gamma.) that adopt
the correct folding and intrachain disulfide bonding patterns (Roy
et al. (1990) J. Biol. Chem. 265:6389-6393). The
A.alpha.B.beta..gamma. trimeric complexes from the Hep G2 cells
associate in pairs to form intact fibrinogen molecules that become
glycosylated and are secreted from the cell (Huang et al. (1993) J.
Biol. Chem. 268:8919-8926).
[0052] Fibrinogen may also be produced in eukaryotic cells that do
not normally synthesize fibrinogen in significant quantities. For
example, eukaryotic cells known to be capable of assembling and
secreting recombinant fibrinogen include baby hamster kidney cells
(BHK), COS cells and Chinese hamster ovary cells (CHO; Roy et al.,
(1991) J. Biol. Chem. 266:4758-4763; Hartwig and Danishefsky (1991)
J. Biol. Chem. 266:6578-6585; Farrell et al. (1991) Biochemistry
30:9414-9420). Methods known to those of skill in the art may be
used to increase the output of recombinant proteins from
transfected cells.
[0053] Within those aspects wherein the first composition further
comprises Factor XIII, it is typically present at a concentration
of from between about 10 U/ml and about 80 U/ml. Factor XIII may be
purified from pooled plasma. Factor XIII may also be produced by a
recombinant process. For example, recombinant Factor XIII may be
produced by host cells such as microbial cells (e.g., yeast cells)
or mammalian cells. Methods for producing recombinant Factor XIII
are disclosed in EP-A-0268772, which is herein incorporated by
reference in its entirety.
[0054] Factor XIII is often co-purified with fibrinogen. For
example, fibrinogen may be purified by chromatography using
DEAE-cellulose and may be further purified by GPRPC-agarose
chromatography (Falls and Farrell (1997) J. Biol. Chem.
272:14251-14256). Factor XIII co-purifies with .gamma.A/.gamma.'
fibrinogen on DEAE-cellulose (Siebenlist et al. (1996) Biochemistry
35:10448-10453), presumably by binding directly to the .gamma.'
chain in .gamma.A/.gamma.' fibrinogen. Factor XIII is depleted from
.gamma.A/.gamma.' fibrinogen purified further on GPRPC-agarose
(Falls and Farrell (1997) J. Biol. Chem. 272:14251-14256).
[0055] The pH of the first composition is usually between about pH
5.0 and about pH 9.0, between about pH 5.5 and about pH 8.5,
between about pH 6.0 and about pH 8.0, between about pH 6.5 and
about pH 7.5, or between about pH 6.7 and about pH 7.2.
[0056] As indicated above, degradation-resistant fibrinogen
sealants employ a second composition comprising thrombin. Thrombin
is usually present in second compositions at a concentration of
between about 4 IU/ml and about 1000 IU/ml, between about 10 IU/ml
and about 150 IU/ml, or between about 15 IU/ml and about 120 IU/ml.
In a particular embodiment, thrombin is present in second
compositions at a concentration of 25 IU/ml, 50 IU/ml, 75 IU/ml, or
100 IU/ml. Thrombin may be purified from, for example, bovine or
human sources. Thrombin may also be produced by a recombinant
process. For example, recombinant thrombin may be produced in
mammalian cells, such as CHO cells. Processes for the production of
thrombin are disclosed in U.S. Pat. Nos. 5,476,777; 5,502,034 and
5,572,692 which are herein incorporated by reference in their
entirety.
[0057] In those embodiments wherein calcium is present in the
second composition, the concentration of calcium may be between
about 1 mM and about 70 mM, between about 20 mM and about 60 mM, or
between about 30 mM and about 50 mM.
[0058] In some embodiments, a second composition of a
degradation-resistant fibrinogen sealant may also contain human
albumin, mannitol, and/or sodium acetate. The pH of the second
composition is usually between pH 5.0 to pH 9.0, between pH 5.5 to
pH 8.5, between pH 6.0 to pH 8.0, between pH 6.5 to pH 7.5, or
between pH 6.8 to pH 7.2.
[0059] Within certain aspects, degradation-resistant fibrinogen
sealants disclosed herein may further employ one or more fibrin I
and/or fibrin II monomer(s). Thus, for example, fibrin I monomers
and/or fibrin II monomers may be prepared in advance of sealant
application from fibrinogen using, for example a proteolytic enzyme
such as thrombin.
[0060] Fibrin I and fibrin II monomers can be prepared from fibrin
polymer. For example, fibrin polymer can be dissolved using a weak
acid solution and the resulting fibrin monomer can be lyophilized
to a fine powder. The powder can be redissolved in a weak acid and
induced to repolymerize by the addition of an alkali buffer.
Alternatively or additionally, the powdered fibrin monomers can be
dissolved in a chaotropic solution, e.g., urea, to a very high
concentration (e.g., in excess of 150 mg/ml) and induced to
repolymerize by the addition of water.
[0061] As will be recognized, fibrin gel structure can be modified
by many different formulation variables including fibrinogen
concentration, Factor XIII concentration, thrombin concentration,
pH, ionic strength, and additives. For example, a fibrin sealant
may additionally contain, for example, coagulation factors, amino
acids, fibronectin, plasminogen, aprotinin, albumin, heparin,
creatine, sodium citrate, anti-fibrinolytic agents, stabilizers,
antibiotics, antibodies, anti-inflammatory compounds, cytokines,
hormones, interferon, protease inhibitors, steroids, anesthetic,
vitamins, chemotherapeutics, and fibroblastic growth factors, NaCl,
arginine, tranexamic acid, and glycine.
Administration of Degradation-Resistant Fibrinogen Sealants
[0062] The degradation-resistant fibrinogen sealants disclosed
herein may be administered in any manner including, for example,
topically, parenterally, or intravenously. For example, for topical
administration, a solution containing fibrinogen, thrombin or both
may be applied to the tissue. The solution may be applied topically
in any manner including, spraying or dripping the solution onto the
tissue. Typically, the solution is applied in short bursts (0.1-0.2
ml) to produce a thin, even layer. If the hemostatic effect is not
complete, a second layer may be applied. The amount of sealant
required depends upon the area of tissue to be treated and the
method of application.
[0063] Degradation-resistant fibrinogen sealants are applied as two
or more compositions, typically the compositions are applied
simultaneously in approximately equal volumes. Typically, the
compositions are admixed upon application.
[0064] In some embodiments, the solutions may be applied, for
example, using a multi-barreled syringe, (e.g., a double-barreled
syringe), a spray tip (PANTAJECT.RTM. with a spray tip), a spray
catheter (e.g., an ENDOFLEX.RTM. spray catheter with an
ENDOFLEX.RTM. spray tip), a catheter (e.g., a CATHEJECT.TM. dual
lumen endoscopic catheter, a flexible PvB catheter), a cannula (a
CATHEJECT.TM. dual lumen cannula), or a probe (e.g., an
ENDOFLEX.RTM. double-lumen probe).
[0065] Standard surgical techniques for hemorrhagic control,
including suture, ligature, and cautery, may be used prior to the
application of the sealant. Excess blood may be removed from the
site of application, if possible, before applying the fibrin
sealant.
[0066] Fibrinogen may also be administered intravenously. For
example, fibrinogen may be injected intravenously to control
hemorrhage. When a subject is bleeding excessively, for example due
to trauma, it is often necessary to replace their fibrinogen by
injection of cryoprecipitate. Fibrinogen injected intravenously
could be used, for example, as a replacement or adjunct therapy to
cryoprecipitate injection in transfusion medicine.
[0067] The fibrinogen to be administered intravenously may be
administered as a composition. For example, fibrinogen may be
formulated in a saline buffer. The concentration of fibrinogen in
the composition may be 1-100 mg/ml. The concentration of fibrinogen
in the composition may be about 5 mg/ml, 10 mg/ml, 15 mg/ml, or 20
mg/ml.
[0068] Fibrinogen .gamma.A/.gamma.' may comprise between about 0%
and about 100% of the total fibrinogen in a first composition.
.gamma.A/.gamma.' fibrinogen may be present in the first
composition at between about 5% and 90% of the total fibrinogen,
between about 10% and about 80% of the total fibrinogen, between
about 20% and about 70% of the total fibrinogen, or at about 30%,
40%, 50%, or 60% of the total fibrinogen.
[0069] The indications for intravenous administration of fibrinogen
are the same or similar to the indications for injection of
cryoprecipitate. For example, fibrinogen may be administered
intravenously when fibrinogen concentration in the blood plasma
reaches below a certain critical cutoff.
[0070] Degradation-resistant .gamma.A/.gamma.' and/or
.gamma.'/.gamma.' fibrinogen sealants will result in decreased
pathophysiologic sequellae of uncontrolled hemorrhage. A
significantly more stable blood pressure will be maintained, as
well as heart rate. Decreased blood loss will result in decreased
loss of blood cells, resulting in a higher hematocrit. In addition,
improved maintenance of clotting parameters measured in the
activated partial thromboplastin time, prothrombin time, and
thromboelastogram due to decreased loss of clotting factors from
dilution and consumption are expected. Blood chemistries will also
be maintained closer to normal ranges, and resuscitation fluid
(lactated Ringer's solution) use should decrease.
Kit
[0071] The present invention also includes kits for the practice of
the methods of the invention. The kits of the instant invention
include the degradation-resistant fibrinogen sealants and a device
for administering the compositions of the sealant. The first and
second compositions may be provided in separate containers or may
be provided "pre-loaded" in the device for administration. For
example, the first and second compositions may be contained in a
double-barreled syringe within the kit, ready for administration.
Exemplary devices for the administration of the
degradation-resistant fibrinogen sealants of the instant invention
are described hereinabove.
[0072] In a particular embodiment, the kits further comprise at
least one component selected from the group consisting of
instruction material, wound dressing (e.g., without limitation, a
bandages, gauzes, and sponges), sutures, other blood clotting
compounds, wound cleaning agents (e.g. alcohol, saline, and means
of irrigation (e.g., squirt bottle)), tourniquets, pain killers
(e.g., analgesics such as narcotic analgesics (e.g., morphine),
non-narcotic analgesics (e.g., aspirin and acetaminophen), and
narcotic antagonistic analgesics), and antibiotics.
[0073] The following Examples are provided to illustrate the
present invention, but are not meant to limit the invention in any
way.
Example 1
Purification of .gamma.A/.gamma.' and .gamma.A/.gamma.A
Fibrinogen
[0074] Plasminogen-free human plasma fibrinogen (Calbiochem) was
dissolved in 39 mM Tris-PO.sub.4, pH 8.6, containing 5 mM
6-aminocaproic acid (EACA) and 0.2 mM phenylmethylsulfonyl fluoride
(PMSF) and dialyzed into the same buffer at 4.degree. C. Insoluble
residue was removed by centrifugation at 10,000.times.g for 30 min
at 4.degree. C. The .gamma.A/.gamma.' and .gamma.A/.gamma.A forms
of fibrinogen were separated using DEAE-cellulose (Finlayson et
al., (1960) J. Clin. Invest. 39:1837-1840). Briefly, the fibrinogen
solution was adsorbed to a column of DEAE-cellulose (6.times.20 cm)
and eluted with a 1200-ml exponential gradient generated in a
600-ml constant volume mixing chamber from the starting buffer (39
mM Tris-PO.sub.4, pH 8.6; 5 mM EACA; and 0.2 mM PMSF) to the final
buffer (193 mM Tris-PO.sub.4, pH 4.6; 5 mM EACA; and 0.2 mM PMSF).
The absorbance was monitored at 280 nm, and 11-ml fractions were
collected. The elution profile showed two peaks; .gamma.A/.gamma.A
fibrinogen composed the first peak and .gamma.A/.gamma.' fibrinogen
composed the second smaller peak.
[0075] .gamma.A/.gamma.' and .gamma.A/.gamma.A may be further
purified using a glycine-L-proline-L-arginine-L-proline-L-cysteine
(GPRPC)-agarose affinity resin (Farrell and Thiagarajan (1994) J.
Biol. Chem. 269:226-231). Briefly, the resin may be prepared by
reacting 10 mg of glycine-L-proline-L-arginine-L-proline-L-cysteine
peptide (Howard Hughes Medical Institute Biopolymer Laboratory,
Seattle, Wash.) with 10 ml of 5-thio-2-nitrobenzoate-agarose
(Pierce) according to the manufacturer's protocol. The dialyzed
.gamma.A/.gamma.' or .gamma.A/.gamma.A fibrinogen pool from
DEAE-cellulose is adsorbed to a column (3 ml) of GPRPC-agarose,
washed with 100 mM NaCl, 50 mM Tris-PO.sub.4, pH 7.8, 5 mM EACA,
0.2 mM PMSF, and then washed with 2 M NaBr, 50 mM Tris-PO.sub.4, pH
7.8, 5 mM EACA, 0.2 mM PMSF. The fibrinogen is eluted in 1-ml
fractions with 2 M NaBr, 20 mM citrate, pH 5.3, 5 mM EACA, 0.2 mM
PMSF and immediately neutralized with 0.02 volume of 2M Tris-HCl,
pH 8.0. The fibrinogen fractions are dialyzed into 137 mM NaCl, 2.7
mM KCl, 10 mM HEPES, pH 7.4, 1 mM CaCl.sub.2 and stored at
-70.degree. C.
Example 2
Preparation of a .gamma.A/y.degree. Fibrin Sealant
[0076] A first solution containing .gamma.A/.gamma.' fibrinogen may
be prepared from .gamma.A/.gamma.' fibrinogen purified as described
in Example 1. The .gamma.A/.gamma.' fibrinogen may be treated with
aluminum hydroxide gel to adsorb the Vitamin K dependent clotting
factors. The .gamma.A/.gamma.' fibrinogen may additionally or
alternatively be incubated with a solvent detergent (SD) mixture
consisting of 1% tri-n-butyl phosphate and 1% Triton X-100 for
inactivation of enveloped viruses. The solvent detergent reagents
may then be removed by castor oil extraction and reverse phase
chromatography, for example a C-18 column, and the preparation may
be treated by pasteurization.
[0077] Prior to pasteurization, sucrose (1.8 g/g column filtrate)
and glycine (0.11 g/g) are added as stabilizers and the mixture is
warmed to 37.degree. C. under stirring. The pH is adjusted to
6.8-7.4. The solution is heated to 60.degree..+-.0.5.degree. C. and
maintained at that temperature for 10 hours.
[0078] After pasteurization, the stabilizers used for heat
treatment are removed by diafiltration and the product is
concentrated by ultrafiltration. An affinity chromatography step is
then used to remove plasminogen from the product, after which it is
concentrated, formulated and sterile filtered. The filtered
solution is filled aseptically in 1 ml, 2 ml, or 5 ml aliquots,
frozen at .ltoreq.-60.degree. C. and stored at -30.degree.
C..+-.5.degree. C. until use.
[0079] A second solution containing thrombin may be produced from
cryo-poor plasma. The cryo-poor plasma is applied to an anion
exchange column for binding to prothrombin and activation into
thrombin. The resultant thrombin does not bind to the column and is
eluted with calcium chloride. Thrombin may then be subjected to SD
treatment for 6 to 6.5 hours at 26.degree. C..+-.1.degree. C. The
SD reagents may be removed by cation exchange chromatography.
Mannitol (as a 15% solution), and human albumin may be added to the
product as stabilizers to a final concentration of 2% (w/w) and
0.2% (w/w), respectively. The stabilized solution may then be
passed through a nanofiltration module.
[0080] The filtrate is formulated with calcium chloride to 40 mM
and the concentration of human albumin is adjusted to 0.6%. The
thrombin bulk solution is sterile filtered and aseptically filled
in 1 ml, 2 ml, or 5 ml aliquots, frozen at .ltoreq.-60.degree. C.,
and stored at -30.degree. C..+-.5.degree. C. until use.
Example 3
Swine Injury Model to Evaluate .gamma.A/.gamma.' Fibrinogen
Sealant
[0081] Degradation-resistant .gamma.A/.gamma.' and
.gamma.A/.gamma.A fibrinogen sealants were tested in a randomized
prospective, blinded study comparing blood loss after an aortic
injury in pigs receiving three different treatments--either
standard CROSSEAL.TM. fibrin sealants (positive control),
.gamma.A/.gamma.' fibrinogen sealants, or albumin (negative
control). The aortic injury model produces reproducible clot
failure following aortic injury.
[0082] A hole was made in the aorta of pigs with a 2.0 mm skin
biopsy punch. The punch was removed and bleeding was initiated.
After a 15 minute initial hemorrhage, animals were assigned
randomly to a fibrinogen sealant treatment group--CROSSEAL.TM.,
degradation-resistant .gamma.A/.gamma.' fibrinogen sealants, or
albumin control. One of the two types of fibrinogen sealants or
albumin was sprayed on the aortic wound using a CROSSEAL.TM. fibrin
applicator (American Red Cross). The investigators were blinded as
to the identity of the sealant. A gauze dressing was applied to
cover the wound using manual pressure. If hemostasis was
incomplete, up to two more applications of sealant and dressing
were applied where necessary with two minutes of manual compression
as described above. Following application of the sealant,
resuscitation was initiated with a 37.degree. C. lactated Ringer's
solution at 250 ml/minute with a roller pump. Mean arterial,
systolic, and diastolic blood pressures and heart rate are recorded
at 10 second intervals throughout the study period using a
continuous data collection system. The mean arterial pressure at
which re-bleeding occurs, the volume of lactated Ringer's solution
required, and the time to re-bleeding were measured. At 60 minutes,
surviving animals were killed by an overdose of a commercially
available euthanasia solution (Beuthanasia) administered at 1 ml/10
lb i.v. Following completion of the study, intra-abdominal blood
loss was measured. Proportions of animals surviving the 60 minute
study period were compared. Primary outcome variables were mean
arterial bleeding pressure at which re-bleeding occurs, blood loss,
mortality, time to death, extent of coagulopathy, resuscitation
requirements and acidosis.
[0083] As shown in FIG. 1, degradation-resistant .gamma.A/.gamma.'
fibrinogen sealant of the present disclosure allowed increased
arterial pressure without hemorrhage when arterial pressure is
restored as compared to albumin or CROSSEAL.TM. fibrin sealant.
Example 4
Percentage of .gamma.A/.gamma.' Fibrinogen Affects Rate of
Fibrinolysis
[0084] Microtiter plate fibrinolysis assays were carried out as
described previously by Jones and Meunier using 96-well assay
plates (Corning 25-880-96; Jones and Meunier (1990) Thromb.
Haemostasis 64:455-463). Fibrinogen and Lys-plasminogen
(Calbiochem) were added to an interim mixing plate containing assay
buffer (0.1 M NaCl, 30 mM NaHCO.sub.3, 4 mM KCl, 1 mM CaCl.sub.2, 1
mM Na.sub.2HPO.sub.4, 0.3 mM MgCl.sub.2, 0.4 mM MgSO.sub.4, 10 mM
HEPES, pH 7.4, 0.01% Polysorbate 80). A separate assay plate
contained .alpha.-thrombin and tissue plasminogen activator
(Calbiochem) in assay buffer. The fibrinogen/plasminogen solution
was then dispensed from the interim plate into the assay plate
wells containing thrombin and tissue plasminogen activator. The
final concentrations of reagents were 1.25 mg/ml fibrinogen, 30
.mu.g/ml Lys-plasminogen, 16 ng/ml tissue plasminogen activator,
and 13.2 NIH units/ml thrombin in a total volume of 100 .mu.l. For
assays containing Factor XIII, Factor XIII was added to a final
concentration of either 10 or 100 .mu.g/ml to the wells in the
interim plate containing the plasminogen/fibrinogen mixture. In
some assays 1 mM N-ethylmaleimide was also added to the interim
plate. The turbidity of the clot was measured at room temperature
every 6 minutes at 405 nm. The optical density was converted to
percent lysis as follows:
% lysis=(A.sub.405.times.100%)/(A.sub.405 maximum)
and plotted versus concentration of .gamma./.gamma.' fibrinogen
(.gamma.A/.gamma.' Fbg). See FIG. 2.
[0085] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
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